Development of a validated UPLC–MS/MS method for PK/PD analysis of SYL930 and its two major metabolites in dogs

A sensitive and specific UPLC‐MS/MS method was developed and validated for the simultaneous determination of 2‐amino‐2‐(2‐(4′‐(2‐propyloxazol‐4‐yl)‐[1,1′‐biphenyl]‐4‐yl)ethyl)propane‐1,3‐diol (SYL930), phosphorylated metabolite (SYL930‐P) and hydroxylated metabolite (SYL930‐M) in dog blood using SYL927 and SYL927‐P, analogues of SYL930, as the internal standards. Analytes were extracted with protein precipitation followed by chromatographic separation on a ZorbaxSB‐C₁₈ column (3.5 μm, 2.1 × 100 mm) with a gradient elution of methanol–water containing 0.1% formic acid (v /v). A triple quadrupole tandem mass spectrometer operating in the positive electrospray ionization mode was used to detect SYL930, SYL930‐P, SYL930‐M and IS transitions of 381.2 → 364.2, 461.2 → 334.2, 397.3 → 380.3, 367.1 → 350.4 and 447.5 → 320.2, respectively. The linear calibration curves for SYL930, SYL930‐P and SYL930‐M were 0.5–500, 0.2–100 and 0.5–100 ng/mL, respectively (r ² > 0.99). The intra‐day and inter‐day precisions (RSD, %) of analytes did not exceed 9.16% except for low QCs (≤16.22%), and the accuracy (RE, %) ranged from −14 to 11.4%. The mean recoveries for SYL930, SYL930‐P and SYL930‐M in dog blood were 85.13–107.94, 73.84–80.08 and 85.64–95.44%, respectively. The validated method was successfully applied to pharmacokinetic and PK/PD studies of SYL930 and its two major metabolites in dogs after an oral administration of SYL930.     

Morphologies,interfacial interaction and mechanical performance of super-tough nanostructured PK/PA6 blends

Nanostructured polyketone (PK)/polyamide 6 (PA6) blends can be readily prepared via melt blending technologies and exhibit ultra-high toughness when PA6 is present as the nanoscale phase domains. When PA6 content is 30 vol%, the impact strength of the blends increases from 21.4 kJ/m² of pure PK to 103.2 kJ/m². The impact strength of the PK/PA6 blends with a 5:5 composition ratio reaches as high as 113 kJ/m². The strong intermolecular force between PK and PA6 molecular chains enables the PA6 nanophase to cavitate to dissipate a significant amount of impact energy and effectively prevents the crack propagation or even terminates the cracks. The fracture mechanism of the PK/PA6 blends was further examined by the essential work of fracture method which proves that PK/PA6 blends show improved ability to prevent crack propagation. This work may deepen the understanding of polymer blend systems with strong hydrogen bonding interaction.     

Bioanalytical strategies for developing highly sensitive liquid chromatography/tandem mass spectrometry based methods for the peptide GLP-1 agonists in support of discovery PK/PD studies

Highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methods have been developed and implemented for the quantitative determination of a number of peptides under evaluation in our Glucagon-Like Peptide-1 (GLP-1) discovery program for the treatment of diabetes. These peptides are GLP-1 receptor agonists. Due to the high potency, low dose, and low exposure of these peptides, LC/MS/MS-based methods with Lower Limits of Quantitation (LLOQs) (low picomolar range) were required to support discovery pharmacokinetic/ pharmacodynamic (PK/PD) studies. Compared with small molecules, many of these peptides posed significant bioanalytical challenges in the development of highly sensitive methods because of their parent signal splitting as a result of the formation of multiply charged states, the unfavorable fragmentation patterns for Selected Reaction Monitoring (SRM) transitions due to the generation of a large number of small mass product ions with relative low intensities, and adsorption issues observed during sample preparation. This paper details the strategies developed to maximize the sensitivity and improve LLOQs from aspects of mass spectrometry, chromatography, and sample preparation. A LLOQ of 10 picomolar was achieved for all of the investigated peptides using 100 μL of mouse plasma. This is a 100-fold improvement on LLOQs over generic LC/MS/MS-based methods when the same sample volume and the same mass spectrometer platform were used. The methods have been implemented in the support of discovery PK/PD studies.     

Simultaneous extraction and determination of antibiotics in soils using a method based on quick,easy, cheap,effective, rugged,and safe extraction and liquid chromatography with tandem mass spectrometry

The increasing use of antibiotics has caused substantial environmental problems, which are a matter of great concern. The aim of this work was to develop a quick, easy, cheap, effective, rugged, and safe method for 20 antibiotic residues in soil. The developed method is based on extraction with acetonitrile and phosphate buffer, clean up with dispersive solid‐phase extraction adsorbent using primary secondary amine, octadecylsilane, followed by liquid chromatography with tandem mass spectrometry determination. We optimized different extraction methods and the ratio of cleanup adsorbents to achieve good recoveries at seven spiking levels that ranged from 61.4 to 118.9% with a relative standard deviation below 20% (n  = 5). The method quantification limit was in the range of 2–5 μg/kg for most analytes. Good linear regression coefficients greater than 0.990 were obtained. This method was applied for the analysis of real agricultural soil samples, confirming the feasibility of the method.     

A novelty strategy for the fast analysis of sulfonamide antibiotics in fish tissue using magnetic separation with high‐performance liquid chromatography–tandem mass spectrometry

A simple, fast and low‐cost extraction method with high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) determination was developed on sulfonamide antibiotics (SAs) in fish tissue. Magnetic separation was first introduced into the rapid sample preparation procedure combined with acetonitrile extraction for the analysis of SAs. Partitioning was rapidly achieved between acetonitrile solution and solid matrix by applying an external magnetic field. Acetonitrile solution was collected and concentrated under a nitrogen stream. The residue was redissolved with 1‰ formic acid aqueous solution and defatted with n‐hexane before analysis. The recoveries of SAs were in the range of 74.87–104.74%, with relative standard deviations <13%. The limits of quantification and the limits of detection for SAs ranged from 5.0 to 25.0 μg ^kg?1 and from 2.5 to 10.0 μg ^kg?1, respectively. The presented extraction method proved to be a rapid method which only took 20 min for one sample preparation procedure. Copyright © 2016 John Wiley & Sons, Ltd.     

Liquid chromatography/electrospray ionization tandem mass spectrometry for the quantification of mitiglinide in human plasma: validation and its application to pharmacokinetic studies

A sensitive and specific method was developed and validated for the determination of mitiglinide in human plasma using liquid chromatographic separation with electrospray ionization tandem mass spectrometric detection. Acidified plasma samples were extracted with ethyl acetate. The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C(18) column with a mobile phase of methanol-10 mm ammonium acetate solution at a flow rate of 0.3 mL/min. Analytes were detected with an Agilent 6410 Triple qudrupole mass spectrometer equipped with an electrospray ionization source in positive multiple reaction monitoring mode: m/z 316.2 (precursor ion) to 298.2 (product ion) for mitiglinide and m/z 318.2 (precursor ion) to 120.2 (product ion) for the internal standard. This method was validated over a linear range of 0.5-4000 ng/mL for mitiglinide in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/mL, while a relative standard deviation (RSD) was less than 3.9%. The intra- and inter-run precision (as RSD, %) obtained from three validation runs were all less than 15%. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies.     

QuEChERS-高效液相色谱-串联质谱法同时测定豆芽中40种植物生长调节剂、杀菌剂、杀虫剂和抗生素类药物残留北大核心CSCD

研究从豆芽质量安全监管的实际需求出发,建立了QuEChERS-高效液相色谱-串联质谱同时测定豆芽中植物生长调节剂、杀菌剂、杀虫剂和抗生素类等40种药物残留的方法。首先通过参数优化确定最佳质谱条件,然后比较不同提取溶剂(甲醇、乙腈、0.1%氨水乙腈、1%乙酸乙腈)、提取方法(超声、振荡)以及乙二胺-N-丙基硅烷化硅胶(PSA)、C_(18)净化剂添加量时40种药物的回收率,确定最优前处理过程。样品用10 mL 1%乙酸乙腈提取两次,超声波辅助提取,100 mg C_(18)为净化剂。选用Waters ACQUITY UPLC BEH C_(18)色谱柱(100 mm×2.1 mm,1.7μm)进行分离,甲醇和0.01%甲酸水为流动相梯度洗脱,多反应监测(MRM)模式进行质谱监测,基质匹配外标法定量。结果表明,40种药物可在15 min内完成色谱分离,在2~200μg/L的线性范围内均呈现出良好的线性关系,相关系数(r^(2))均大于0.99,检出限(LOD)和定量限(LOQ)分别为0.1~3μg/kg和0.3~9μg/kg。以阴性豆芽为基质,分别在5、10、50μg/kg 3个水平下进行加标回收试验,40种药物的平均回收率为78.5%~115.3%,相对标准偏差(RSD)为1.3%~9.7%(n=6)。将该方法用于分析邯郸本地豆芽中40种药物的污染状况,结果显示4-氯苯氧乙酸、6-苄基腺嘌呤、多菌灵、2,4-二氯苯氧乙酸(2,4-D)、赤霉素和恩诺沙星检出率较高,分别为28.6%、19.0%、9.5%、9.5%、4.8%和4.8%,含量范围为37.5~352.4、32.4~273.1、28.8~38.7、16.1~20.2、19.9和13.6μg/kg。研究建立的方法简单、快速,灵敏度高,适用于大批量豆芽中40种药物的快速准确测定。     

A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of echinacoside and its pharmacokinetic application in rats

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of echinacoside in rat plasma was established and fully validated. A single step of liquid–liquid extraction with n‐butanol was utilized. Chromatographic separation of the analyte and the internal standard (IS), chlorogenic acid, from the sample matrix was performed using a Capcell‐MG C₁₈ analytical column (100 2.0 mm × 5 µm), with a gradient of acetonitrile and water containing 0.1% acetic acid as the mobile phase. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source operated in negative ion selected reaction monitoring mode. The method was linear in the concentration range 10–2500 ng/mL. The deviations of both intra‐ and inter‐day precisions (RSD) were 7.1% and the assay accuracies were within 99.2–106.5%. Echinacoside proved to be stable during sample storage, preparation and analysis when an antioxidant solution was used. The method was successfully applied to a pharmacokinetic study in rats after an intragastric administration of echinacoside (100 mg/kg). With the lower limit of quantification at 10 ng/mL, this method proved to have sufficient selectivity, sensitivity and reproducibility for the pharmacokinetic study of echinacoside. Copyright © 2009 John Wiley & Sons, Ltd.     

A sensitive method for determination of furanodiene in rat plasma using liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

Furanodiene, a sesquiterpene component extracted from the essential oil of the rhizome of Curcuma wenyujin Y.H. Chen et C. Ling (Wen Ezhu), is widely used in traditional Chinese medicine. A sensitive analytical method was established and validated for furanodiene in rat plasma, which was further applied to assess the pharmacokinetics of furanodiene in rats receiving a single dose of furanodiene. Liquid chromatography tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring mode was used in the method and costundide was used as internal standard. A simple protein precipitation based on methanol was employed. The simple sample cleanup increased the throughput of the method substantially. The method was validated over the range of 1–1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1 ng/mL for furanodiene in plasma. Intra‐ and inter‐day accuracies for furanodiene were 88–115 and 102–107%, and the inter‐day precision less than 14.4%. After a single oral dose of 10 mg/kg of furanodiene, the mean peak plasma concentration of furanodiene was 66.9 ± 23.4 ng/mL at 1 h, the area under the plasma concentration–time curve (AUC_{0–10 h}) was 220 ± 47.8 h ng/mL, and the elimination half‐life was 1.53 ± 0.06 h. After an intravenous adminstration of furanodiene at a dosage of 5 mg/kg, the area under the plasma concentration–time curve was 225 ± 76.1 h?ng/mL, and the elimination half‐life was 2.40 ± 1.18 h. Based on this result, the oral bioavailability of furanodiene in rats at 10 mg/kg is 49.0%. Copyright © 2011 John Wiley & Sons, Ltd.     

A validated liquid chromatographic/tandem mass spectrometric method for the determination of phencyclidine in microliter samples of rat serum

A liquid chromatographic/tandem mass spectrometric method is described for the determination of phencyclidine (PCP) in small volumes of rat serum (e.g. 50 microl). Samples were extracted using a mixed-mode strong cation-exchange column and then separated isocratically using a narrow-bore (2.1 mm i.d.) 3 microm Hypersil phenyl column and a mobile phase consisting of an ammonium formate buffer (pH 2.7) with 60% (v/v) methanol. Detection was accomplished using positive ion electrospray ionization in the multiple reaction monitoring mode. Mass spectra were obtained and peaks were observed at an m/z (% abundance) of 244 (100), 159 (25), and 86 (89). Tandem mass spectra were also obtained from the m/z 244 precursor ion with peaks observed at m/z 159 (100), 86 (96), and 91 (11). Optimum serum PCP sensitivity and precision were obtained at a transition of m/z 244 --> 159. Matrix-associated ion suppression did not significantly affect the accuracy (100-112%) or precision (CV < or =8%) of the assay. The lower limit of quantitation was 1 ng ml(-1) in 50 microl of serum. The method was used to study the serum pharmacokinetics of PCP in rats after an intravenous bolus dose of PCP.     

Liquid chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study

A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed for the determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile and internal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandem mass spectrometry by the multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear over the concentration range from 0.3 to 1000 ng/mL. The lower limit of quantification was 0.3 ng/mL using 50 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively. The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of different methods for the determination of several quinolonic and cinolonic antibiotics in trout muscle tissue by HPLC with fluorescence detection

Summary Quinolonic and cinolonic derivatives are mainly used as antibacterials in fish-farms. In this paper we describe a careful revision of the treatment procedures of samples and a prodedure for the determination of residues of these compounds. Because of the complexity and duration of these procedures, several studies have been carried out and these have lead to a simpler and shorter method. Three approaches have been examined: lyophilization followed by extraction with chloroform, solid-liquid extraction with chloroform and solid-liquid extraction with sodium hydroxide solution, followed by liquid-liquid partition in chloroform. Some previous studies into the partition equilibrium are also included. As a result of our studies we propose a procedure with a lower number of steps than those previously described in the literature. This method has been applied to the analysis of nalidixic, 7-hydroxymethylnalidixic and oxolinic acids and cinoxacin in trout muscle. These analysis have been carried out using an HPLC system equipped with a C₁₈ column and fluorimetric detection. The mobile phase was acetonitrile:oxalic acid. The recoveries obtained were: 70–97% for 7-hydroxymethylnalidixic acid, 75–78% for nalidixic acid, 71–95% for oxolinic acid and 72–85% for cinoxacin.     

Performance evaluation of elemental analysis/isotope ratio mass spectrometry methods for the determination of the D/H ratio in tetramethylurea and other compounds--results of a laboratory inter-comparison

Tetramethylurea (TMU) with a certified D/H ratio is the internal standard for Site-specific Natural Isotope Fractionation measured by Nuclear Magnetic Resonance (SNIF-NMR) analysis of wine ethanol for detection of possible adulterations (Commission Regulation 2676/90). A new batch of a TMU certified reference material (CRM) is currently being prepared. Whereas SNIF-NMR has been employed up to now, Elemental Analysis/Isotope Ratio Mass Spectrometry ((2)H-EA-IRMS) was envisaged as the method of choice for value assignment of the new CRM, as more precise (better repeatable) data might be obtained, resulting in lower uncertainty of the certified value. In order to evaluate the accuracy and intra- and inter-laboratory reproducibility of (2)H-EA-IRMS methods, a laboratory inter-comparison was carried out by analysing TMU and other organic compounds, as well as some waters. The results revealed that experienced laboratories are capable of generating robust and well comparable data, which highlights the emerging potential of IRMS in food authenticity testing. However, a systematic bias between IRMS and SNIF-NMR reference data was observed for TMU; this lack of data consistency rules out the (2)H-IRMS technique for the characterisation measurement of the new TMU CRM.     

High-performance isotope-labeling liquid chromatography mass spectrometry for investigating the effect of drinking Goji tea on urine metabolome profiling

Urine is a human biofluid that is widely used for metabolomics research on disease biomarker discovery.Ideally,the metabolome profiles generated from comparative groups of individuals should mainly consist of the endogenous human metabolites that reflect the healthy states of the individuals.However,external factors,such as diet,may alter the urine metabolome profile by either introducing a significant amount or variety of exogenous metabolites to urine or inducing changes of the metabolome profile.Thus,strict control of the external factors during the sample collection process is critical for urine metabolomics aimed at discovery of disease biomarkers.In this work,we describe a study to determine the effect of drinking Goji tea,which is considered a nutritional supplement drink in some regions of the world,on urine metabolome profile.The purpose of this work is not to determine the nutritional values of Goji tea,but to investigate whether drinking a moderate amount of Goji tea 1-3 h(short-term effect)or 12 h(longer-term effect)before urine collection can cause significant variations of urine metabolome profiles.A highly sensitive dansylation isotope labeling liquid chromatography mass spectrometry(LC-MS)method was used to determine the urine metabolomes before and after drinking Goji tea.From the studies of the short term(<3 h)and longer term(12 h)effects of drinking Goji tea,it is clear that the consumption of a moderate amount of Goji tea does not affect the urine metabolome significantly.Fasting for 12 h should be sufficient to remove any potential interference of Goji metabolites from the human urine metabolome profile.     

Application of a UPLC‐MS/MS method for the analysis of alosetron in human plasma to support a bioequivalence study in healthy males and females

A simple, rapid and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for the determination of alosetron (ALO) in human plasma. The assay method involved solid‐phase extraction of ALO and ALO 13C‐d3 as internal standard (IS) on a LichroSep DVB‐HL (30 mg, 1 cm³) cartridge. The chromatography was performed on an Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 µm) column using acetonitrile and 2.0 mm ammonium formate, pH 3.0 adjusted with 0.1% formic acid (80:20, v/v) as the mobile phase in an isocratic mode. For quantitative analysis, the multiple reaction monitoring transitions studied were m/z 295.1/201.0 for ALO and m/z 299.1/205.1 for IS in the positive ionization mode. The method was validated over a concentration range of 0.01–10.0 ng/mL for ALO. Post‐column infusion experiment showed no positive or negative peaks in the elution range of the analyte and IS after injection of extracted blank plasma. The extent of ion‐suppression/enhancement, expressed as IS‐normalized matrix factor, varied from 0.96 to 1.04. The assay recovery was within 97–103% for ALO and IS. The method was successfully applied to support a bioequivalence study of 1.0 mg alosetron tablets in 28 healthy Indian male and female subjects. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the quantification of methyl protodioscin in rat plasma: application to a pharmacokinetic study

A high performance liquid chromatography/tandem mass spectrometry assay was first developed and validated for the quantification of methyl protodioscin (MPD), a natural furostanol saponin with distinct antitumor activity, in rat plasma with 17alpha-ethinylestradiol as internal standard (IS). Methanol-mediated protein precipitation was employed for plasma sample pretreatment. The separation was achieved on a C(18) column (150 x 4.6 mm, i.d., 5 microm) by isocratic elution with methanol-water (72:28, v/v) as mobile phase at a flow rate of 1.0 mL/min. Ion acquisition was performed in selective reaction monitoring positive mode by monitoring the transition of m/z 1085.7 --> 1053.7 for MPD, and in selective ion monitoring negative mode by monitoring the deprotonated ion m/z 295.5 for IS. The assay was linear over the concentration range of 2.024-270.0 microg/mL with 2.024 microg/mL as the lower limit of quantification. It was specific, accurate, precise and reproducible with intra- and inter-run RSD <8.3% and RE between -11.5 and 12.8%. The assay was successfully applied to a preclinical pharmacokinetic study after an intravenous dose of 40 mg/kg MPD to rats.     

Quantitative LC/MS/MS method and in vivo pharmacokinetic studies of vitexin rhamnoside, a bioactive constituent on cardiovascular system from hawthorn

A simple and accurate liquid chromatography coupled with tandem mass spectrometry method was developed for determination and in vivo pharmacokinetic studies of vitexin rhamnoside in rat plasma. After protein precipitation using methanol, the analytes were separated by a Luna C(18) column with an isocratic elution and analyzed by mass spectrometry in multiple reaction monitoring mode using the respective negative ion at m/z 577.2-293.0 for vitexin rhamnoside and m/z 593.2-413.0 for internal standard (IS) vitexin glucoside. The method was validated systematically within the concentration range 5-5000 microg/L (R > 0.996) and the lower limit of quantitation was 5 microg/L. Acceptable precision and accuracy were acquired for concentrations over the standard curve range. It was further applied to assess pharmacokinetics and bioavailability of vitexin rhamnoside after intravenous and oral administration to rats. The oral bioavailability of vitexin rhamnoside was only 3.57%, which indicated that vitexin rhamnoside had poor absorption or underwent extensive first-pass metabolism. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

A robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of 5‐azacytidine

The DNA methyltransferase inhibitor 5‐azacytidine is being evaluated clinically as an oral formulation to treat various solid tumors. A sensitive, reliable method was developed to quantitate 5‐azacytidine using LC‐MS/MS to perform detailed pharmacokinetic studies. The drug of interest was extracted from plasma using Oasis MCX ion exchange solid‐phase extraction 96‐well plates. Chromatographic separation was achieved with a YMC J'sphere M80 C₁₈ column and isocratic elution with a methanol–water–formic acid (15:85:0.1, v/v/v) mobile phase over a 7 min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5‐azacytidine. The assay range was 5–500 ng/mL and proved to be accurate (97.8–109.1%) and precise (CV ≤ 9.8%). Tetrahydrouridine was used to stabilize 5‐azacytidine in blood/plasma samples. With the addition of tetrahydrouridine, long‐term frozen plasma stability for 5‐azacytidine at ?70°C has been determined for at least 323 days. The method was applied for the measurement of total plasma concentrations of 5‐azacytidine in a cancer patient receiving a 300 mg oral daily dose. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatography/tandem mass spectrometry method for quantification of trans-stilbene glycoside in rat plasma and its pharmacokinetic application

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of trans‐stilbene glycoside (SG) in rat plasma. As trans‐SG can be rapidly isomerized under light exposure, trans‐SG plasma samples were prepared in the dark and assayed immediately. Trans‐SG and internal standard were extracted by protein precipitation. Chromatographic separation was achieved on a C₁₈ column with a gradient elution program. The detection of analytes was performed by negative ion via multiple reaction monitoring mode. The precursor‐to‐product ions of m/z 405.1 → 242.9 for trans‐SG and m/z 389.1 → 226.9 for polydatin (internal standard) were monitored. No interference of endogenous components was observed for any plasma samples that we studied.The method was linear over the concentration range of 1.0–1000.0 ng/mL with a good correlation coefficient. The lower limit of quantification was 1.0 ng/mL for trans‐SG. The intra and inter‐batch accuracy for trans‐SG in stable rat plasma samples ranged from 93.3 to 102.7% and the variation was less than 8.1%. The extraction recoveries of trans‐SG in rat plasma were from 102.8 to 112.4% and the matrix effects were also acceptable. This method was successfully applied to pharmacokinetic study of trans‐SG in rats after intravenous administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of an Orally Bioavailable,Brain-Penetrant Compound with Selectivity for the Cannabinoid Type 2 Receptor

Modulation of the endocannabinoid system (ECS) is of great interest for its therapeutic relevance in several pathophysiological processes. The CB2 subtype is largely localized to immune effectors, including microglia within the central nervous system, where it promotes anti-inflammation. Recently, a rational drug design toward precise modulation of the CB2 active site revealed the novelty of Pyrrolo[2,1-c][1,4]benzodiazepines tricyclic chemotype with a high conformational similarity in comparison to the existing leads. These compounds are structurally unique, confirming their chemotype novelty. In our continuing search for new chemotypes as selective CB2 regulatory molecules, following SAR approaches, a total of 17 selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs were synthesized and tested for their ability to bind to the CB1 and CB2 receptor orthosteric sites. A competitive [³H]CP-55,940 binding screen revealed five compounds that exhibited >60% displacement at 10 μM concentration. Further concentration-response analysis revealed two compounds, 4k and 4q, as potent and selective CB2 ligands with sub-micromolar activities (K_i = 146 nM and 137 nM, respectively). In order to support the potential efficacy and safety of the analogs, the oral and intravenous pharmacokinetic properties of compound 4k were sought. Compound 4k was orally bioavailable, reaching maximum brain concentrations of 602 ± 162 ng/g (p.o.) with an elimination half-life of 22.9 ± 3.73 h. Whether administered via the oral or intravenous route, the elimination half-lives ranged between 9.3 and 16.7 h in the liver and kidneys. These compounds represent novel chemotypes, which can be further optimized for improved affinity and selectivity toward the CB2 receptor.